TNBC Progression Research Articles

A natural antisense lncRNA controls breast cancer progression by promoting tumor suppressor gene mRNA stability.

The human genome encodes thousands of long noncoding RNA (lncRNA) genes; the function of majority of them is poorly understood. Aberrant expression of a significant number of lncRNAs is observed in various diseases, including cancer. To gain insights into the role of lncRNAs in breast cancer progression, we performed genome-wide transcriptome analyses in an isogenic, triple negative breast cancer (TNBC/basal-like) progression cell lines using a 3D cell culture model. We identified significantly altered expression of 1853 lncRNAs, including ~500 natural antisense transcript (NATs) lncRNAs. A significant number of breast cancer-deregulated NATs displayed co-regulated expression with oncogenic and tumor suppressor protein-coding genes in cis. Further studies on one such NAT, PDCD4-AS1 lncRNA reveal that it positively regulates the expression and activity of the tumor suppressor PDCD4 in mammary epithelial cells. Both PDCD4-AS1 and PDCD4 show reduced expression in TNBC cell lines and in patients, and depletion of PDCD4-AS1 compromised the cellular levels and activity of PDCD4. Further, tumorigenic properties of PDCD4-AS1-depleted TNBC cells were rescued by exogenous expression of PDCD4, implying that PDCD4-AS1 acts upstream of PDCD4. Mechanistically, PDCD4-AS1 stabilizes PDCD4 RNA by forming RNA duplex and controls the interaction between PDCD4 RNA and RNA decay promoting factors such as HuR. Our studies demonstrate crucial roles played by NAT lncRNAs in regulating post-transcriptional gene expression of key oncogenic or tumor suppressor genes, thereby contributing to TNBC progression.

Cancer-Associated MORC2-Mutant M276I Regulates an hnRNPM-Mediated CD44 Splicing Switch to Promote Invasion and Metastasis in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most lethal subtype of breast cancer, with a high propensity for distant metastasis and limited treatment options, yet its molecular underpinnings remain largely unknown. Microrchidia family CW-type zinc finger 2 (MORC2) is a newly identified chromatin remodeling protein whose mutations have been causally implicated in several neurologic disorders. Here, we report that a cancer-associated substitution of methionine to isoleucine at residue 276 (M276I) of MORC2 confers gain-of-function properties in the metastatic progression of TNBC. Expression of mutant MORC2 in TNBC cells increased cell migration, invasion, and lung metastasis without affecting cell proliferation and primary tumor growth compared with its wild-type counterpart. The M276I mutation enhanced binding of MORC2 to heterogeneous nuclear ribonucleoprotein M (hnRNPM), a component of the spliceosome machinery. This interaction promoted an hnRNPM-mediated splicing switch of CD44 from the epithelial isoform (CD44v) to the mesenchymal isoform (CD44s), ultimately driving epithelial-mesenchymal transition (EMT). Knockdown of hnRNPM reduced the binding of mutant MORC2 to CD44 pre-mRNA and reversed the mutant MORC2-induced CD44 splicing switch and EMT, consequently impairing the migratory, invasive, and lung metastatic potential of mutant MORC2-expressing cells. Collectively, these findings provide the first functional evidence for the M276I mutation in promoting TNBC progression. They also establish the first mechanistic connection between MORC2 and RNA splicing and highlight the importance of deciphering unique patient-derived mutations for optimizing clinical outcomes of this highly heterogeneous disease.Significance: A gain-of-function effect of a single mutation on MORC2 promotes metastasis of triple-negative breast cancer by regulating CD44 splicing. Cancer Res; 78(20); 5780-92. ©2018 AACR.

Abstract B18: ADSL controls pyrimidine metabolism and triple-negative breast tumorigenesis

Abstract Triple-negative breast cancer (TNBC) represents 15% of all breast carcinomas. The unfavorable prognosis and aggressive biology of TNBC highlight the need to find new targeted therapies. Previous study from our lab has shown that the prolyl hydroxylase EglN2 contributes to TNBC progression, with its depletion leading to a decrease in TNBC cell invasion. However, the underlying mechanism remains elusive. In our current study, we developed a novel enzyme-substrate trapping strategy followed by mass spectrometry and identified adenylosuccinate lyase (ADSL) as a potential EglN2 hydroxylase substrate. EglN2 binds directly with ADSL and catalyzes ADSL hydroxylation on proline residues, which might affect ADSL activity. We demonstrated that ADSL is specifically upregulated in TNBC cell lines and patients. Functionally, depletion of ADSL led to decreased anchorage-independent growth, TNBC cell invasion, and orthotopic tumor growth. Mechanistically, integrated analyses of metabolomics and RNA-seq showed that ADSL controls pyrimidine biosynthesis that is essential for TNBC cell proliferation and invasion. Therefore, our study suggests that EglN2-ADSL signaling axis can serve as a novel therapeutic avenue in TNBC by regulating pyrimidine metabolism. Citation Format: Giada Zurlo, Jeremy Simon, Cheng Fan, Adam Robinson, Javier Rodriguez Martinez, Alex Kriegsheim, Jason Locasale, Charles Perou, Qing Zhang. ADSL controls pyrimidine metabolism and triple-negative breast tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B18.

Abstract 4964: Metabolically activated macrophages mediate obesity-driven TNBC progression

Abstract Triple-negative breast cancer (TNBC) patients have the poor prognosis due to their high metastatic potential and lack of targeted therapies. Emerging epidemiological data suggest that obesity is associated with increased incidence of more aggressive triple-negative breast cancer; however, mechanisms are unclear. During obesity, adipocytes within breast tissues recruit macrophages. M2-like macrophages can exacerbate TNBC progression; however, obese breast tissue is reported to be enriched with M1-like adipose tissue macrophages (ATMs), which are anti-tumorigenic. These results raise a paradox: how does obesity promote tumor progression if it activates an anti-tumorigenic macrophage phenotype? Here we show that obesity promotes a distinct pro-inflammatory phenotype (metabolically activated macrophages; MMe) in breast ATMs, which promotes TNBC progression. We showed that ATMs isolated from obese women express cell surface markers of MMe (CD36, ABCA1) but not M1 (CD38, CD319) macrophages. We further found that pretreating TNBC cells with MMe (but not M1) derived conditioned media increase tumorsphere growth, and frequency of tumor-incidence when injected into mice in limited dilutions. Remarkably, we saw the same effect with obesity on tumor-incidence in both spontaneous and syngeneic TNBC mouse models. Moreover, blocking cytokine expression specifically in MMe using myeloid specific genetic knockout mice diminishes the effect of obesity on tumor-incidence. Interestingly, a subset of these MMe-secreted cytokines shared a common signal transducer, gp130, which led us to investigate the role of gp130 in tumors. Knocking down gp130 in cancer cells inhibits the MMe-induced stat-3 phosphorylation and tumorsphere growth in vitro, and diminishes the MMe-mediated effect of obesity on tumor-incidence in mice. Together these findings suggest that obesity-induced metabolically activated macrophages (MMe) in breast adipose tissue secrete inflammatory cytokines that promotes the TNBC tumor-progression by activating gp130 in cancer cells. A comprehensive understanding of the signaling mechanism(s) involved in metabolic activation of mammary ATMs would enable development of targeted therapies towards this specific pro-tumorigenic macrophage phenotype, thereby leaving the immune system of cancer patients intact. Citation Format: Payal Tiwari, Ariane Blank, Chang Cui, Kelly Schoenfelt, Seema A. Khan, Marsha R. Rosner, Lev Becker. Metabolically activated macrophages mediate obesity-driven TNBC progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4964.

Abstract LB-350: IgG4 expressing B cells associate with poor survival in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is an aggressive form of breast cancer with high levels of lymphocyte infiltration. Although high levels of infiltrating B cells are present in TNBC, their role in progression of this disease is unclear. IgG4 is one of four subclasses of IgG and is the least abundant in healthy human serum. IgG4 has been found at higher percentages in both tissue and serum in cases of chronic inflammation, which promotes class switching to IgG4. Expression and secretion of IgG4 by B cells may be non-beneficial and potentially harmful in the cancer setting. Unlike the other subclasses of IgG, IgG4 does not activate complement dependent cytotoxicity and has a lower affinity for FcγR, thus resulting in a poor ability to engage immune effector cells and activate antibody-depended cell-mediated cytotoxicity. Additionally, IgG4 can compete with other IgG subclasses by binding to antigens and can sequestering IgG through Fc-Fc interactions, thus impairing potential anti-tumor antibody-mediated responses. As IgG4 expressing B cells have been shown to be associated with poor outcomes in other aggressive cancers such melanoma, glioblastoma and pancreatic cancer, we sought to determine the expression of IgG4 in TNBC. We performed immunohistological staining of IgG4 in paraffin-embedded tissue microarrays from 75 treatment-naive TNBC specimens obtained from women who underwent surgical resection of their tumors at the Helen F Graham Cancer Center and Research Institute from 2006-2007. We found that 69 % of TNBC cases contain IgG4+ cell infiltration. Moreover, the presence of IgG4+ cells is significantly correlated with poorer progression free (p<0.0003) and overall survival (p<0.0001) as determined by a Log-rank (Mantel-Cox) test. These findings indicate the presence of an immunosuppressive B cell population. Further study is needed to characterize these cells and to determine the significance of IgG4+ tumor infiltrating B cells, and their potential immunosuppressive role in the progression of TNBC. Citation Format: Nicole Flynn, Ugochukwu Amadi, Rajasekharan Somasundaram, Jennifer Sims-Mourtada. IgG4 expressing B cells associate with poor survival in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-350.

Abstract 2100: Using in vitro models to identify newly synthesised proteins involved in the progression of breast cancer

Abstract Triple negative breast cancer (TNBC) is a distinct subtype of breast cancer defined by tumours deficient in the expression of the three main immuno-histochemical markers: the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth receptor 2 (HER2). Although accounting for only 15-20% of total breast cancer, TNBC is characterized by an aggressive phenotype and poor prognosis. As a result, it is the cause of a disproportionate amount of breast cancer deaths, with a higher incidence amongst younger women and ethnic minorities. In addition, lack of ER, PR and HER2 expression means there are no specific targeted therapies available. Significant efforts are being made to improve detection and survival rates for women affected by this disease. In order to achieve this, there is an urgent need to identify novel biomarkers and molecular targets so that we can more quickly and accurately diagnose breast cancer and develop a better understanding of disease progression in patients diagnosed with TNBC. Genomic studies of patients with TNBC have shown that there is strong heterogeneity within the sub-type and a need for more precise classification at a molecular level. One target that has emerged from these studies is the epidermal growth factor receptor (EGFR). The EGFR is found to be over expressed in approximately 50% of TNBC cases and amplification of the EGFR gene is correlated with poor prognosis. Our study aims to investigate exactly how the progression of TNBC is influenced by specific micro-environmental cues and identify novel targets involved in the growth and migration of tumour cells. We are investigating this target by developing cell culture based, in vitro models of EGFR driven cancer metastasis. Using Click-iT chemistry and mass spectrometry analysis, and by culturing the cells in both 2D and 3D in vitro models, we have identified proteins that are newly synthesised by the cancer cells as they are stimulated to migrate towards epidermal growth factor (EGF). Our approach has generated a list of proteins that span a myriad of different functions known to be important in cancer progression such as cell metabolism, intracellular calcium sensing, anti-oxidation and regulation of cell structure. We hypothesise that these newly synthesised proteins play a vital role in cancer cell migration and metastasis. Interaction and molecular function analysis of this list using STRING has shown an enrichment in calcium ion binding proteins. We are particularly interested the role of these calcium-regulated proteins in influencing the structure and behaviour of migrating cells. Currently, we are investigating the role of several of these proteins in regulating the cells response to the tumour microenvironment. Directing pharmacological interventions towards these newly synthesised proteins has the potential to target invasive cancer cells and provide a highly specific and effective therapy for TNBC. Citation Format: Amira F. Mahdi, Beatrice Malacrida, Kieran McGourty, Aoife J. Lowery, Patrick A. Kiely. Using in vitro models to identify newly synthesised proteins involved in the progression of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2100.

Abstract B50: The role of CXCL13-CXCR5 signaling in triple-negative breast cancer progression

Abstract Triple-negative breast cancer (TNBC) is an aggressive cancer with poor prognosis and is difficult to treat. There is a major health disparity associated with TNBC; the number of fatalities associated with this disease is significantly higher in African American women compared to their Caucasian counterparts. The mechanisms behind these differences have not been entirely elucidated. There are no targeted therapies for TNBC; instead, standard therapy for this disease includes a combination of chemotherapeutic drugs: doxorubicin (DOX), paclitaxel (TAX), and/or cyclophosphamide (CTX). Even more troubling, the efficacy of standard TNBC therapies is marginal and patients eventually become resistant to this intervention. TNBC patients suffer from chemoresistance and metastasis and ultimately succumb to this disease. Understanding TNBC and its health disparity requires a better understanding of the race-specific differences in breast tumor biology, molecules leading to breast inflammation, and the mechanisms associated with TNBC chemoresistance. Chemokines and their receptors play a significant role in TNBC. Our laboratory was the first to show that CXCL13-CXCR5 signaling mediates prostate cancer cell growth, migration, invasion, and survival (1-3). We also demonstrated that CXCL13-CXCR5 signaling induces cancer progression signaling pathways PI3K, AKT, ERK, and Jun (1;4). These preliminary and published findings provide the rationale or scientific premise in support of our hypothesis that CXCL13-CXCR5 signaling promotes TNBC progression migration, invasion, proliferation, and chemoresistance. To determine whether CXCL13-CXCR5 signaling induces TNBC, the expression profile of CXCL13 and CXCR5 RNA, associated miRNAs, and cancer progression genes (epithelial mesenchymal transition [EMT] markers) will be correlated with disease-free and overall survival of TNBC patients in silico using RNA-seq datasets from the Sequence Read Archive (SRA). Fastq data set files were downloaded from SRA, checked for quality control, aligned, and normalized using open-source platforms. Hierarchical clustering, gene set enrichment analysis, disease progression analysis, and statistics were conducted using Partek Genomic Suite. Pathway enrichment analysis was conducted using Ingenuity Pathway Studios. Overall, CXCL13-CXCR5 signaling is implicated in TNBC progression, migration, and invasion. Citation Format: Courtney D. Dill, Adaugo Okoro, Dongquan Chen, James W. Lillard, Jr.. The role of CXCL13-CXCR5 signaling in triple-negative breast cancer progression [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr B50.

Abstract LB-125: Atovaquone suppresses triple negative breast tumor growth by inhibiting myeloid derived suppressor cells

Abstract Triple negative breast cancer (TNBC) is considered to be the most aggressive and malignant neoplasm and is highly metastatic in nature. Metastatic TNBC is difficult to treat thus leading to extremely poor survival. The major contributing factor in TNBC progression and resistive nature is its ability to evade the immune surveillance. Modulating the immune system in breast tumors could be an important strategy for cancer therapeutics. The present study evaluates the immune response of atovaquone, an antiprotozoal drug in various TNBC cell lines and its efficacy in three independent in vivo models. In the first experiment, HCC1806 human breast cancer cells were orthotopically implanted in mammary fat of female mice. Oral administration of atovaquone reduced HCC1806 breast tumor growth by 45%. On the other hand, oral administration of atovaquone inhibited the growth of highly aggressive CI66 breast tumors by 70%. Paclitaxel is the first line chemotherapeutic agent for metastatic breast cancer. In third in vivo experiment, 4T1 paclitaxel resistant (4T1R) cells were injected in mice and atovaquone suppressed paclitaxel resistant tumor growth by 42%. Myeloid derived suppressor cells (MDSCs) are known to suppress immune function. Our current results demonstrated 70% and 30% suppression of tumor and blood MDSCs respectively, with atovaquone treatment in mouse bearing HCC1806 tumors. We also observed about 25% reduction in MDSCs with atovaquone treatment in mice with CI66 as well as 4T1R tumors. MDSCs induce regulatory T cells (Treg cells) which are immunosuppressive. Our results showed reduction of Treg cells by 12% and 17% with atovaquone treatment in HCC1806 tumors and in 4T1R tumors respectively. In addition, decrease in TGF-beta (13%) and IL-10 (40%) in HCC1806 tumor lysates was observed by atovaquone, an evidence of reduction in immunosuppressive cytokines produced by MDSCs. It appears that the mechanism of immune suppression of atovaquone by MDSCs in our model may be through TGF-β and IL-10, which are potent immunosuppressive cytokines and induce Treg cells that allow cancer cells to escape from immune surveillance. This is the first report demonstrating that immune modulation by atovaquone treatment is associated with breast tumor growth suppression, prompting further investigation to establish atovaquone as a treatment option for TNBC patients. Citation Format: Nehal Gupta, Stephen Wright, Sanjay Srivastava. Atovaquone suppresses triple negative breast tumor growth by inhibiting myeloid derived suppressor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-125.

PO-487 Prognosis of triple negative breast cancer is associated with MHC II genes

IntroductionTriple negative breast cancer (TNBC) is clinically to denote women with invasive breast cancer whose tumours lack expression of oestrogen receptor (ER-), progesterone receptor (PR-), or overexpression of HER2/Neu. TNB tumours behave aggressively and are not candidates for ER or HER2/Neu targeted therapy. In previous published study of 47 TNBC patients with RNA-seq data, we discovered that twenty-four genes related MCH pathway exhibited significantly higher expression in tumour tissue from patients who did not relapse, including eleven genes representation of the MHC II pathway. In this study, we further investigated 24 genes in MCH pathway in prognosis of TNBC. This work will lead in future guidance in treatment of TNB based on patient’s’ gene profile and also provides means to assess prognosis in TNBC.Material and methodsForty-seven snap frozen primary TNBC tumour specimens were analysed using RNA-seq. Descriptive analysis and logistic regression model were used to identify genes that can predict TNB good prognosis (no relapsed). Odds ratio and 95% two-sided confidence intervals were presented. The optimal cut point of expression level was determined from the receiver operating characteristic analysis and sensitivity and specificity were estimated. This study has approval of Institutional Review Board.Results and discussionsMCH II genes CD74 and CIITA had significantly association with TNBC progression free survival. When TNBC patients had high CIITA, they were 10 times likely to have no relapsed than patients with low expression (odds ratio OR=10.8, 95% CI 2.8–41.9, p=0.006). The sensitivity and specificity is 75% and 78.3% respectively; TNB who had higher expression of CD74 had 3 times more likely to have no relapsed (OR=3.1, 95% CI 0.95–10.28, p=0.0609) with sensitivity and specificity of 62.5% and 65.2% respectively. We used multivariable approach with all 24-gene panel and identified three genes, CIITA, CTSH and KRT14 together can predict no relapsed with 87.5% sensitivity 78.3%. The accuracy is 83% if TNBC had high expression of CIITA, CTSH and KRT14. With a total of 47 patients, we predicted 17 out 22 relapsed, and 21 out 25 no relapsed correctly. The results was validated using the public microarray data from 199 patients with TNBC confirmed.ConclusionHigher expression of genes in MCII pathway, e.g. CIITA, CD74, not only associated with TNBC progression free survival, they also have high accuracy to predict TNBC patient’s no relapse regardless of age, race, and tumour grade and tumour stage.

Abstract 2002: Ganoderma lucidum extract (GLE) reduces stem cell markers in triple-negative breast cancer models via STAT3 regulation

Abstract Triple-negative breast cancer (TNBC) is a subtype of cancer described by the lack of estrogen receptor, progestogen receptor, and HER2 receptor. It accounts for approximately 15-20% of all breast cancer (BC) cases worldwide. TNBC aggressiveness might be explained by the presence of BC stem cells (BCSCs), which are a subpopulation of cancer cells involved in tumor initiation, metastasis, and therapy resistance. Interestingly, the signal transducer and activator of transcription 3 (STAT3) participates in the development and progression of BCSCs, but its role in TNBC progression remains unclear. Moreover, BCSCs can be identified by the CD44+/CD24- and ALDH+ phenotype, which correlates with a worse prognosis in TNBC patients. Furthermore, CSCs can be characterized based on the expression of transcription factors involved in stemness (Nanog, Sox2, and Oct4A), where activation of STAT3 leads to enhanced Nanog, Oct4 and Sox2 expression. Importantly, a major challenge arises to develop nontoxic targeted therapies to improve the outcome of TNBC patients. In this regard, our laboratory investigates the role of Ganoderma lucidum extract (GLE), a medicinal mushroom commonly used in complementary and alternative medicine. We hypothesize that GLE will decrease stem cell properties via STAT3 regulation in TNBC cells. Our in vitro results demonstrate that GLE significantly downregulates STAT3 and JAK2 in SUM-149 and MDA-MB-231, TNBC cell lines. In addition, GLE significantly decreases the secretion of IL-6 at 24hr in MDAMB231 cells. Further flow cytometry results show that GLE significantly decreases the CD44+/CD24- population at 24hr and 72hr in MDA-MB-231 cells. In this regard, GLE significantly decreases the expression of ALDH at 24hr and 72hr in MDAMB231 cells. Moreover, GLE decreases the expression of Nanog at 24hr, and all three transcription factors at 72hr. Additionally, our in vivo studies demonstrate that GLE significantly decreases tumor volume of mice injected with CD44+/CD24- sorted cells of the MDA-MB-231 cell line. Finally, tumor weight of GLE treated mice was marginally significantly lower (p=0.053) compared to vehicle. We can conclude that GLE decreases stemness of TNBC via STAT3 regulation. Citation Format: Tiffany J. Rios, Alicia Marti, Gabriela Ortiz, Michelle M. Martínez-Montemayor. Ganoderma lucidum extract (GLE) reduces stem cell markers in triple-negative breast cancer models via STAT3 regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2002.

Abstract 1900: Disruption of EGFR and integrin b1 signaling in triple-negative breast cancer through targeting SHC1

Abstract Triple-negative breast cancer (TNBC) accounts for 15-20% of breast cancer cases and is more prevalent among young women and African American women. SHC1 (Src Homology 2 Domain Containing Transforming Protein 1) is an adaptor protein that can transmit cell surface receptor signaling and activate the Ras-ERK pathway. SHC1 is found to be overexpressed in breast cancers and is important in the regulation of tumor progression and drug resistance in ER+ breast cancer. Elevated SHC1 correlates with poor prognosis in breast cancer patients. However, the biological significance of SHC1 in TNBC is not known. In this study, we performed siRNA-mediated knockdown to determine the importance of SHC1 in cell proliferation and drug response in TNBC. We found that although siSHC1 slightly inhibited the tumor growth of TNBC cells, siSHC1 + paclitaxel significantly decreased cell proliferation and increased caspase-3/7 activity in vitro. In invasion assays, siSHC1 significantly decreased the invasion of TNBC cells. To better understand the systemic regulation by SHC1, proteomic analysis was performed after siSHC1 or Scr transfection. Pathway analysis revealed the involvement of PI3K/AKT, FAK, and actin cytoskeleton pathways, by disruption of EGFR and integrin β1 signaling, in the control of TNBC progression and drug sensitivity. In tumor explant studies, siSHC1 significantly enhanced paclitaxel-induced tumor shrinkage and reduced lung metastasis. Our data suggest that SHC1 is a potential therapeutic target in TNBC. Citation Format: Ya-Fang Chang, Elizabeth Cordell, Yuhao Wang, Geoffrey Greene. Disruption of EGFR and integrin b1 signaling in triple-negative breast cancer through targeting SHC1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1900.

Abstract 2541: Combined androgen and glucocorticoid receptor (AR/GR) activity drives TNBC progression

Abstract Background: A recent effort to distinguish early-stage TNBC based on outcome and to expose driver pathways using gene profiling/cluster analysis divided TNBC into four subtypes- basal-like, mesenchymal, immunomodulatory, and luminal androgen receptor (LAR). The clinical AR+ TNBC subtype (>10% AR by IHC) comprises up to 35% of metastatic disease. The discovery of this TNBC subtype and the concomitant development of potent new anti-androgens for prostate cancer have led to several clinical trials evaluating AR antagonists in TNBC. In parallel, glucocorticoid receptor (GR) expression and activation have been shown to mediate anti-apoptotic gene expression in TNBC. However, to our knowledge, no one has examined the coordinate expression of GR and AR activity and the potential crosstalk downstream of dual GR and AR activation. We hypothesized that AR/GR cooperative gene expression pathways may induce TNBC therapy resistance and tumor progression. Methods and Results: We analyzed GR+ TNBC primary tumor gene expression from TCGA samples and found a wide range of AR expression. To examine whether GR-associated gene expression differed in high versus low AR+ TNBC, we divided TCGA TNBC primary tumors (N=123) at the AR median into “high” versus “low” AR expression. We then identified relative GR target gene expression levels in those genes identified from a previous TNBC meta-analysis. Invasion, motility and proliferation pathways were found to be most significantly expressed among GR-associated genes in AR high TNBC (top 5 significant pathways). Similarly, we found that GR and AR co-activation in AR+ MDA-MB-453 TNBC cells significantly increased cell migration compared to GR or AR activation alone. In addition, when MDA-MB-453 cells were exposed to the AR antagonist enzalutamide (enza) for more than 30 days (long-term, LT), the magnitude of increase in cell migration following GR activation increased, suggesting that increased GR activity may compensate for long-term AR blockade. Furthermore, long-term enza exposure (LTenza) in AR+ MFM-223 and MDA-MB-453 cells resulted in increased GR mRNA and protein levels. The magnitude of GR-mediated target gene expression was also significantly increased in LTEnza versus control-treated cells. In vivo, LTenza-treated MDA-MB-453 xenografts were relatively insensitive to paclitaxel chemotherapy treatment, consistent with increased GR activity compared to control cells. Conclusions: We conclude that coordinate GR and AR expression in TNBC contributes to a particularly poor outcome in TNBC. Downstream AR/GR gene expression favoring cell survival and invasive phenotypes may contribute to this outcome. Consideration of dual AR/GR inhibition in TNBC may increase the effectiveness of anti-androgen therapy. Citation Format: Deniz N. Dolcen, Eva Tonsing-Carter, Ryan Harkless, Caroline Kim, Kathleen Bowie, Gini Fleming, Suzanne Conzen. Combined androgen and glucocorticoid receptor (AR/GR) activity drives TNBC progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2541.

TGF-beta/AURKA pharmacologic targeting to halt stemness and TNBC progression.

e13127Background: TGF-b signaling promotes an invasive TNBC phenotype through activation of epithelial to mesenchymal transition (EMT) reprogramming. Breast cancer cells that undergo EMT acquire a ...

Abstract P6-06-04: Targeting replication stress in triple negative breast cancer treatment regimen: An emerging approach

Abstract Triple-negative breast cancers (TNBCs) represent aggressive heterogeneous subtype of breast cancer with poor clinical outcome. TNBCs have been reported to have high levels of replication stress due to i) various oncogene activations (C-myc or EGFR) ii) germline BRCA mutations iii) “BRCAness” in the absence of BRCA mutations in sporadic TNBCs. Replication stress is known to cause genomic instability, promote tumorigenesis and plays a critical role in therapy resistance in TNBCs. Therefore, targeting replication stress has emerged as an effective approach for better TNBC treatment through further downregulation of the remaining checkpoints to induce catastrophic failure of TNBC cells proliferation. Herein, we evaluated the anticancer efficacy of Carbazole Blue (CB), a synthetic analogue of Carbazole, on TNBC cells growth and progression. Our results demonstrated that CB inhibits short and long term viability of TNBC (MDA-MB-231, MDA-MB-468 and BT549) cells in a dose dependent manner without affecting normal mammary epithelial (MCF-10A) cells. In addition, CB treatment significantly reduced proliferation of TNBC cells, as evidenced by the BrdU proliferation assay. Consistent with this, our results further demonstrated that CB treatment induced G1/S cell cycle arrest and apoptosis in TNBCs. Importantly, systemic delivery of CB using nanoparticle-based delivery approach suppressed breast cancer growth without inducing toxicity, in preclinical orthotopic xenograft and PDX mouse models of TNBC. Furthermore, our gene microarray analysis revealed that CB treatment modulates the expression and activity of several genes known to be involved in DNA replication (CDC6, CDT1, MCMs, Claspin, POLE and PCNA) and associated DNA repair machinery such as (XRCC3, Exo1 and RAD51), which play pivotal roles in replication stress. Our results for the first time highlight the potential use of CB as a novel and potent therapeutic agent for treating TNBCs. As exploiting replication stress to treat cancer is gaining major interest, compound/s that may induce replication stress and inhibit DNA repair ability of cancer cells, has immense translational potential. Citation Format: Rajamanickam S, Park JH, Bates K, Timilsina S, Eedunuri VK, Onyeagucha B, Subbarayalu P, Abdelfattah N, Jung KH, Favours E, Mohammad TA, Chen H-IH, Vadlamudi RK, Chen Y, Kaipparettu BA, Arbiser JL, Rao MK. Targeting replication stress in triple negative breast cancer treatment regimen: An emerging approach [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-06-04.

Rutin as A Novel c-Met Inhibitory Lead for The Control of Triple Negative Breast Malignancies

ABSTRACTTriple negative breast cancer (TNBC) has high metastatic and mortality potential and lacks effective and selective therapeutic options. Aberrant dysregulation of the receptor tyrosine kinase c-Met promotes TNBC progression, motility and survival and therefore considered a valid therapeutic target. Among various identified anticancer agents, plant polyphenols (PPs) including flavonoids, have been shown to be safe and proven for their antitumor activity through modulating diverse macromolecular targets. This study reports the bioassay-guided identification of the common flavonol glycoside rutin as breast cancer cell proliferation, migration and invasion inhibitor. The cell free Z′-LYTE kinase assay, Western blot and in silico docking experiments uncovered, for the first time, c-Met kinase as a potential mechanistic target for rutin-mediated anticancer effects on TNBC cell lines. Likewise, the intraperitoneal injection of rutin at 30 mg/kg, 3X/week, significantly reduced the growth of the TNBC MDA-MB-231/GFP orthotopic xenograft in nude mouse model. These results clearly designate the functional dietary flavonoid rutin as a potential lead for the prevention and control of c-Met-dependent breast malignancies.

Abstract 3249: Thymoquinone inhibits elongation factor 2 kinase signaling axis by inducing tumor suppressor miR-603 in triple negative breast cancer cells

Abstract Triple negative breast cancer (TNBC), is a highly heterogeneous and aggressive subtype of breast cancer (BC), which poses a significant clinical challenge. TNBC constitutes about 15-20 % of BC cases, and is characterized by lack of estrogen (ER), progesterone (PR) and human epidermal growth factor 2 (HER2) receptors. Thus, patients cannot benefit from targeted therapies such as anti-estrogens (eg.Tamoxifen) and anti-HER2 (eg.trastuzumab) treatments. Therefore, identification of new molecular targets and treatment strategies are highly warranted to improve patient outcome. We previously reported that elongation factor 2 kinase (EF-2K) is highly expressed in TNBC cell lines and is associated with poor patient survival and prognosis. Furthermore, in vivo targeting of EF-2K by siRNA nano-therapeutics inhibited cell proliferation, migration/invasion, and significantly decreased tumor growth in 2 different orthotopic TNBC mouse models (MDA MB-231 and MDA MB-436). Collectively, our work suggests that EF-2K is an excellent novel therapeutic target in TNBC. In search of a potential safe and effective EF-2K inhibitor, we identified Thymoquinone (TQ), a dietary natural compound, known to have diverse anti-cancerous properties in several in vitro and in vivo models, including TNBC. However, the mechanism by which TQ mediates its effects are not well elucidated. Our current study is the first to demonstrate that TQ inhibits protein and mRNA expression of EF-2K, as well as its clinically significant downstream targets such as Src/FAK, PI3K/AKT, and CyclinD1; resulting in a significant decrease in proliferation, migration/invasion of TNBC cells. To determine the molecular mechanism by which TQ inhibits EF-2K expression, we investigated if TQ induces tumor suppressor microRNAs that we identified to bind to the 3’-UTR of EF-2K. We found that TQ induces miR-603 expression, which we had reported to directly bind and inhibit EF-2K expression; resulting in decreased TNBC growth and progression, both in vitro and in vivo. Furthermore, we showed that inhibition of nuclear factor kappa B (NF-κB) (a well established target for TQ), also induces miR-603 and inhibits EF-2K expression in TNBC cells. In conclusion, our study is the first to show that TQ treatment decreases EF-2K expression through modulating the NF-κB/miR-603 axis; ultimately resulting in decreased cell proliferation, migration/invasion of TNBC. Our data suggests a novel molecular mechanism for TQ that represents a potential therapeutic strategy in inhibiting TNBC tumor growth and progression. Citation Format: Nashwa N. Kabil, Recep Bayraktar, Nermin Kahraman, Bulent Ozpolat. Thymoquinone inhibits elongation factor 2 kinase signaling axis by inducing tumor suppressor miR-603 in triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3249. doi:10.1158/1538-7445.AM2017-3249

Abstract 2251: Tumor tissue gene expression in association with survival of triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is an aggressive type of cancer with limited treatment options. Previous studies have shown that gene expression profiles were associated with TNBC prognosis. Information on specific genes that are predictive of TNBC outcome is limited. Using data and samples from a cohort of 469 TNBC cases from Shanghai Breast Cancer Survival Study (SBCSS), we systematically evaluated expressions of 302 genes in tumor tissue with survival of TNBC. Genes included in the study are PAM50 genes, genes encoding drug metabolizing enzymes and, genes implicated in TNBC biology and progression based on literature reviews. Gene expression levels were measured in total RNA isolated from formalin-fixed paraffin-embedded breast cancer tissues using the NanoString nCounter assay. Cox regression was applied to evaluate disease-free survival (DFS) and overall survival (OS) in association with gene expression. Analysis was adjusted for known predictors of TNBC outcome, including age, disease stage, basal like subtype and DUSP4 gene expression. During a median follow-up of 5.3 years (range: 0.7-8.9 years), 100 deaths and 92 recurrences/breast cancer deaths were documented. Expression levels of 17 genes were significantly associated with OS, and 15 genes with DFS (P<0.05). The top 5 most significant genes are EOMES (Hazard Ratio, HR = 0.88; 95% Confidence Interval, 95% CI: 0.82-0.96), GZMK (HR = 0.91, 95% CI: 0.85-0.97), TUBB3 (HR = 1.12, 95% CI: 1.03-1.21), SIT1 (HR = 0.90, 95% CI: 0.83-0.98) and ZNF224 (HR = 0.89, 95% CI: 0.81-0.97) for OS, and TUBB3 (HR = 1.14, 95% CI: 1.05-1.24), KRT14 (HR = 1.08, 95% CI: 1.02-1.14), BAG1 (HR = 1.19, 95% CI: 1.05-1.35), CD44 (HR = 0.73, 95% CI: 0.58-0.92) and EOMES (HR = 0.89, 95% CI: 0.82-0.97) for DFS. Study is on-going to replicate the findings of this study. Citation Format: Shuyang Wang, Qiuyin Cai, Hui Cai, Pingping Bao, Jie Wu, Fei Ye, Wei Zheng, Ying Zheng, Xiao-Ou Shu. Tumor tissue gene expression in association with survival of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2251. doi:10.1158/1538-7445.AM2017-2251

Abstract LB-266: DAXX promotes de novo lipogenesis in triple-negative breast cancer

Abstract Triple-negative breast cancers (TNBCs), defined by the absence of ER and PR expression and the lack of HER2 amplification, have no targeted treatment options, are highly aggressive, and exhibit poor prognosis. Identification of drivers and associated signaling pathways, particularly for TNBCs, is needed to assist the development of targeted therapies, to prolong disease-free survival, and to improve clinical outcome for cancer patients. Using high-throughput microarray-based global transcriptome profiling, we found that transcriptional regulator DAXX depletion strikingly impaired while DAXX overexpression enhanced the expression of genes in the de novo lipogenesis pathways in TNBCs cell line. Consistently, unbiased global metabolomics and lipidomics profiling by Ultra-High performance Liquid Chromatography coupled High-Resolution Mass Spectrometry (UHPLC-HRMS) also revealed that DAXX promotes de novo lipogenesis in both TNBC cell line as well as in corresponding xenograft tumors. In analyzing large number of genomic datasets including The Cancer Genome Atlas (TCGA), MolEcular Taxonomy of BReast cancer International Consortium (METABRIC), and The Cancer Cell Line Encyclopedia (CCLE) dataset, we found that DAXX expression is significantly higher in TNBC tumor samples and cell lines, and also DAXX gene expression positively correlates with that of key lipogenesis enzymes. The role of DAXX in cancer metabolism is previously unknown. Our integrated OMICs data analysis thus indicates that DAXX promotes de novo lipogenesis in TNBC. The mechanism through which DAXX regulates lipid metabolism is being investigated in our laboratory. Overall, our study identifies a potentially important pathway driving TNBC progression and suggests potential therapeutic targets for treating this aggressive form of breast cancer that still lacks curative treatment options. Citation Format: Iqbal Mahmud, Timothy J. Garrett, Daiqing Liao. DAXX promotes de novo lipogenesis in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-266. doi:10.1158/1538-7445.AM2017-LB-266

Abstract 4229: Metabolically active macrophages: A novel link between obesity and TNBC

Abstract Triple negative breast cancer (TNBC) patients have an extremely poor prognosis due to their high metastatic potential and lack of targeted drug therapies. Emerging epidemiological data suggest that obesity is strongly linked to the incidence and severity of TNBC. However, mechanisms by which obesity potentiates TNBC progression are unclear. Adipose tissue macrophages (ATMs) are an attractive mechanistic link between obesity and TNBC because they are the predominant type of macrophage in the breast during early tumorigenesis and obesity causes accumulation of ATMs. However, during obesity, the macrophage phenotype was reported to switch from an apparent pro-tumorigenic M2-like phenotype to an apparent anti-tumorigenic M1-like phenotype. This result raises a paradox: how does obesity promote tumor progression if it activates an anti-tumorigenic macrophage phenotype? Here we show that metabolic dysfunction promotes a mechanistically distinct pro-inflammatory phenotype (metabolic activation; MMe) in breast adipose tissue macrophages isolated from obese women. These ATMs express cell surface markers of MMe (CD36, ABCA1) but not M1 (CD38, CD319, CD274) macrophages. We further demonstrate that pretreating TNBC cells with conditioned media derived from MMe but not M1 macrophages promotes mammosphere formation as well as invasion in vitro, and intravasation of cancer cells into the blood in vivo. Moreover, pre-treated cancer cells showed a two-fold increase in the expression of stem-like cell markers (OCT4, SOX2 and NANOG). Remarkably, we saw the same effect on ‘stemness’ of tumor cells in a spontaneous TNBC mouse model when fed a 60% high fat diet and a significant increase in tumor incidence in obese mice compared to lean mice in a syngeneic mouse model. We further show that blocking inflammatory cytokines expression in MMe(s) using genetic knockouts inhibits the ability of MMe(s) to promote both mammosphere formation and invasion. These findings suggest that obesity-induced changes to mammary adipose tissue reprogram macrophages to an MMe phenotype that potentiates TNBC initiation and metastasis via inflammatory cytokines. A comprehensive understanding of the signaling mechanism(s) involved in metabolic activation of mammary ATMs would enable development of directed therapies towards this specific pro-tumorigenic macrophage phenotype, thereby leaving the immune system of cancer patients intact. Citation Format: Payal Tiwari, Kelly Schoenfelt, Ariane Blank, Chang Cui, Marsha Rich Rosner, Lev Becker. Metabolically active macrophages: A novel link between obesity and TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4229. doi:10.1158/1538-7445.AM2017-4229

Abstract P4-15-03: Regulation of miRNA-29c and its gene targets in preneoplastic progression of triple negative breast cancer

Abstract P4-15-03: Regulation of miRNA-29c and its gene targets in preneoplastic progression of triple negative breast cancer